Methicillin resistant S. aureus has posed major public health problems worldwide. The basis of methicillin resistance in S. aureus strains is the functional replacement of innate penicillin binding proteins (PBPs) with alternative penicillin binding protein PBP2a which binds poorly to most 2-lactams. A major dilemma in the diagnosis of methicillin resistant S. aureus infections is the time required for diagnosis after hospital admission, thus enabling intra-hospital spread of these dangerous pathogens. Conventional cultures for the diagnosis of methicillin resistant S. aureus take at least two days. A newer and faster method for diagnosis is the PCR-based test. Although this method is faster than conventional cultures, the assay entails expensive equipment and higher per-unit cost. Therefore, there is a gap in the rapid diagnosis of methicillin-resistant S. aureus which requires a simple standalone assay with a faster turnaround time and at a cheaper cost. Saureus Inc. is a company that focuses on enabling technology for rapid diagnosis of important healthcare- related pathogens. Taking advantage of our knowledge in Gram+ molecular genetics, we have engineered L. lactis bacteria to enable heterologous expression of Spa, a surface protein of S. aureus. As Spa binds efficiently to mammalian IgGs, we demonstrated that rabbit anti-PBP2a antibodies bind at a high affinity to the recombinant Spa anchored on the surface of L. lactis. To facilitate rapid cell lysis, lysostaphin, an enzyme highly specific for lysis of S. aureus (much less so for S. epidermidis), will be deployed in a way that only lyses S. aureus efficiently, resulting in spillage of intracellular and membrane-bound contents including PBP2a. Several epitope-specific rabbit anti-PBP2a monoclonal antibodies immobilized on the surface of L. lactis bacteria will agglutinate with PBP2a to result in a clumping reaction that can be easily visualized without any extraneous visual aid. This assay is simple and cost effective and does not rely on operator skill or sophisticated lab equipment; these characteristics are typically absent in PCR-based assays. Accordingly, we plan to develop two specific aims for this proposal: I) establishing the agglutination platform based on rabbit monoclonal antibodies to PBP2a immobilized on Spa-expressing L. lactis and the lysing platform based on lysostaphin-mediated release of cellular contents from MRSA; II) optimization of variables that would enhance sensitivity, specificity, reagent stability and ease of use for the lead prototype agglutination kit for MRSA. Upon completion of these studies, we plan to apply for Phase II SBIR funding for studies on clinical samples of S. aureus. Successful conclusion of clinical studies will justify an application to the FDA-CLIA for approval of the diagnostic kit.